ABSTRACT (Overall Component) The three Projects vigorously pursue an understanding of antiviral CD4 and CD8 T cells, linked by the theme: what transcription factors regulate these cells and how do they do so? T cell differentiation into various effector cells, and the capacity to differentiate into memory cells, are important parts of adaptive immunity to pathogens and cancers. Transcription factors (TFs) are central regulators of these differentiation processes. The identification of key TFs regulating different pathways of CD4 and CD8 T cell differentiation have been central to understanding the biology of these cells. However, it is abundantly clear that TFs do not act in isolation and many TFs may be important inducers or repressors of a T cell differentiation pathway. The biggest challenge to studying TF network biology is that experimental manipulation of more than 1 factor at a time under controlled conditions has not been generally feasible, particularly in primary cells in vivo. Therefore, the focus on 1 gene at a time has been an experimental necessity for decades; the generation of double and triple knockout mice is excessively time consuming. Therefore, our approach as a group to this serious problem has been focused on experimental approaches whereby we can modulate and test many genes in parallel for their roles in antiviral T cell responses in vivo, using custom shRNAmir vector-based approaches. We have established these systems, and are able to perform genetic screens, in vivo, in primary CD4 or CD8 T cells, probing differentiation and function. Our projects propose a highly integrated approach to revealing the biology of regulation and differentiation of T follicular helper (Tfh) CD4 T cells, memory CD4 T cells, CTL CD8 T cells, circulating memory CD8 T cells, and tissue-resident CD8 and CD4 T cells (Trm). The three PIs have worked intensively together over the past 5 years to develop important insights into TF biology in T cells. The interactions are not simply via shared cores, they involve extensive shared experiments and ideas regarding TFs, CRs, epigenetics, and gene regulation networks. This is most clearly seen in our 2017 Nature paper on Trm and Runx3. That work is connected to our earlier work together predicting TF regulators of gene expression in T cells, and our most recent paper together, defining the dominant pioneer factor role of Runx3 in the earliest steps of effector CD8 T cell differentiation and defining how Runx3 interacts with other TFs that we have studied, including T-bet, Blimp-1, IRF4, TCF1, and Id2. Our collective experience is that our screens identify multiple critical factors, more than can be comprehensively studied in the context of a single project. Therefore, the equally important challenge?and where our Program?s synergy stands out?is narrowing down the candidate molecules to particular ?high value? TFs and CRs that have a major influence on T cell programming. Our three Projects acquire such data in the orthogonal screens and epistasis experiments proposed by each Project, which allow us to converge quickly on important targets to study in detail.